The invention relates to the use of a specific affinity existing between two molecules to purify or identify another molecule.
There currently exists a variety of methods, materials, and approaches for the separation of a particular protein from the other components of a biological sample. One general approach exploits the non-specific affinity of a protein for a substrate. For example, proteins may be separated based upon their molecular charge using ion exchange chromatography. In ion exchange chromatography, protein mixtures are applied to an oppositely charged, chromatographic matrix (e.g., DEAE- or carboxymethyl-Sephadex), and the various proteins bind to the matrix by reversible, electrostatic interactions. The adsorbed proteins are eluted, in order of least to most strongly bound, by increasing the ionic strength or by varying the pH of the elution buffer.
Another general approach makes use of a protein's physical characteristics as a means of separation. For example, a protein may be separated based upon its size, using gel filtration. By this method, protein mixtures are applied to a gel-filtration column containing a chromatographic matrix (e.g., Sephadex, Bio-Gel, or Sephracryl) of defined pore size. Proteins are eluted, generally with an aqueous buffer, collected as individual chromatographic fractions and analyzed.
Both of the general approaches described above suffer from an inability, in most cases, to specifically isolate the protein to be purified from other proteins having similar physical characteristics.
A third general approach makes use of the specific affinity of a protein for a purifying reagent. A protein, for example, may be purified using an antibody specific for that protein. In one common application, the antibody is bound to a column substrate (e.g., Sepharose) and a high molecular weight antigen applied to the column. High molecular weight antigen molecules pass freely into and out of the pores and bind to antibodies covalently attached to the matrix. Bound proteins are then eluted by destabilizing the antigen-antibody complex, e.g., by brief exposure to high pH or low pH buffers. Antibodies are also used to purify proteins by immunoprecipitation. Antigen-antibody complexes may be precipitated following aggregation, or alternatively, antibodies may be covalently linked to Sepharose and immunoaffinity complexes isolated by centrifugation. In either method, the protein of interest is then released from the complex and analyzed, generally by polyacrylamide gel electrophoresis. Immunoprecipitation methods rely on optimizing specific antigen-antibody complex formation while minimizing non-specific interactions; as such, in some instances, the protein of interest is not precipitated; in other instances, the precipitate contains the desired protein significantly contaminated with other proteins. In addition, this method requires that an antibody specific for the protein of interest be available or that it can be prepared.